Use of HIV-1 gp120 and gp160 proteins modified in the V3 loop for the preparation of vaccine compositions and formulations containing the same

ABSTRACT

Use of HIV-1 gp120 and gp160 proteins which have been modified in the V3 loop for preparing vaccine compositions and formulations containing them which induce a systemic and mucosal immunity. Use of a recombinant HIV-1 Env protein, in which the V3 loop is partially or completely deleted, for preparing a vaccine composition which is capable of inducing an immunity which is at the same time humoral, cellular and mucosal with respect to HIV-1. The vaccine composition comprises: a recombinant Env protein as defined above, optionally at least one compound selected from the group consisting of: (1) the vaccination adjuvants selected from the group consisting of derivatives comprising divalent or trivalent ions: aluminium hydroxide or calcium phosphate, and muramylpeptide derivatives and (2) liposomes and optionally at least one pharmaceutically acceptable vehicle.

This is a continuation application of U.S. application Ser. No.09/632,806, filed Aug. 4, 2000, now allowed, which claims the benefit ofFrench Patent Application 0004310, filed Apr. 4, 2000, both of which areherein incorporated by reference in their entirety.

The present invention relates to the use of HIV-1 gp120 and gp160proteins which have been modified in the V3 loop for preparing vaccinecompositions, as well as to formulations containing them which arecapable of producing a humoral, cellular and mucosal immune response.

The type-1 human immunodeficiency virus (HIV-1) is the aetiologicalagent of AIDS. HIV-1 induces a persistent infection in humans whichleads to a severe immunodeficiency. The envelope of HIV is composed oftwo glycoproteins, gp120 and gp41, which are derived from a precursor,gp160, by proteolytic cleavage. Five conserved regions, C1 to C5, andfive variable regions, V1 to V5, have been demonstrated in theglycoprotein of the envelope. Three functional regions play an essentialrole in the first steps of the infection, and have been identified: theCD4-binding site (in the C4 region), the V3 region, which is essentialto infectivity, and finally a very hydrophobic region which is locatedat the N-terminal end of gp41, and which participates in the fusionbetween the membrane of the target cell and the viral envelope. The V3loop is hypervariable, immunodominant and corresponds to the principalneutralizing-antibody-inducing determinant (PND).

It is generally accepted that neutralizing antibodies play an importantrole in protection against viral infection (1, 2).

However, in the case of the type 1 human immunodeficiency virus (HIV-1),the neutralizing antibodies which develop at an early stage of theinfection do not prevent the progression of the disease.

Specifically, in infected individuals, the neutralizing antibodies, whenthey exist, exhibit a very narrow neutralization range and often do notneutralize the strain(s) which infect(s) the patient (3, 4). In the sameway, the neutralizing antibodies induced by vaccination with gp160/120in solution are generally specific for the strain used for the vaccinepreparation and are directed against one or more determinants in the V3loop of gp120 (5).

Although the V3 loop is hypervariable, a Gly-Pro-Gly-Arg (GPGR) (aminoacid residues 1-4 of SEQ ID NO: 1) tetrapeptide which is located at thetop of the loop, as well as two cysteines at its base, are present inalmost all known isolates of HIV-1, which indicates that this sequenceis essential to a vital cycle step of the virus (6).

The roles of the V3 loop (cellular tropism, infectivity, induction ofrestricted-range neutralizing antibodies and role in pathogenesis) havebeen demonstrated (6).

The inventors have modified the env gene of HIV-1 by carrying outdeletions which eliminate or decrease the hypervariable epitopes of theV3 loop. The results, obtained with a partial deletion in the V3 loop ofgp160 while keeping the tip of the V3 loop, i.e. the GPGRAF (SEQ IDNO: 1) sequence and the two cysteines at its base, show that themodified protein ΔV3-GPGRAF is expressed in the same way as theunmodified protein, and that it reacts with an anti-HIV human referenceserum to a degree which is similar to unmodified recombinant gp160 (7).

A protein in which the V3 loop is completely deleted was also produced;it is the protein ΔV3+, which is also expressed and which has amolecular mass compatible with the deletion.

This set of elements has led the inventors to formulate the hypothesisthat the V3 loop would represent a decoy for the immune system, and thatthe modification or elimination of this loop might induce aconfirmational change in the molecule, which would reveal itself by theinduction of a neutralizing activity directed against other epitopeswhich are more conserved but which show weak immunogenicity during thenatural infection or subsequent to a vaccination with the nativeprotein.

Faced with the AIDS epidemic, the development of an anti-AIDS vaccinewhich is capable of halting the propagation of the disease isimperative; indeed, the World Health Organization estimates that in2002, there could be between 50 and 75 million people in the worldinfected with HIV.

The inventors consequently gave themselves the goal of producing avaccine composition which is better at meeting the requirements of theart in that it is capable of inducing a humoral, cellular and mucosalimmune response which exhibits a wide-ranging neutralization due to theinduction of antibodies which are capable of neutralizing various typesof HIV-1 strain, and in particular both laboratory strains and clinicalstrains (primary isolates).

A subject of the present invention is the use of a recombinant HIV-1 Envprotein, in which the V3 loop is partially or completely deleted, forpreparing a vaccine composition which is capable of inducing an immunitywhich is at the same time humoral, cellular and mucosal with respect todivergent strains of HIV-1.

The inventors have now found, surprisingly, that the proteins in whichthe V3 loop is partially or completely deleted are actually capable ofinducing a wide-ranging protective immunity which is at the same timehumoral (neutralizing antibodies), cellular (cytotoxic T lymphocytes)and mucosal (neutralizing secretory IgA productions).

“Wide-ranging immune response or immunity” is intended to mean the setof humoral and cellular factors which protects the body against an HIV-1infection, in accordance with the definition by J. F. Bach (ImmunologyTreaty, Flammarion, 1993).

In accordance with said use, said recombinant Env protein is selectedfrom the group consisting of the Env proteins in which the V3 loop ispartially deleted: ΔV3-GPGRAF recombinant gp160 and gp120 proteins, andthe Env proteins in which the V3 loop is completely deleted: ΔV3+recombinant gp160 and gp120 Env proteins.

A subject of the present invention is also a vaccine composition,characterized in that it comprises:

a recombinant Env protein as defined above,

optionally at least one compound selected from the group consisting of:

(1) the vaccination adjuvants selected from the group consisting ofderivatives comprising divalent or trivalent ions: aluminium hydroxideor calcium phosphate, and muramylpeptide derivatives and

(2) liposomes and

optionally at least one pharmaceutically acceptable vehicle.

According to one advantageous embodiment of said vaccine composition, itcomprises a recombinant Env protein as defined above which is anchoredonto unilamellar synthetic lipid vesicles or liposomes (immunosomes)which comprise a phosphatidylcholine:cholesterol molar ratio of about8:1, and which have a size of between 70 and 150 nm, preferably 90 nm,as described in patent EP 47480.

Such a vaccine composition can advantageously be administered eithergenerally or systemically: orally, parenterally, or locally (via therectal or vaginal route, for example); it is preferably administered viaa route which involves a direct contact with a mucous membrane, andwhich thus makes it possible to obtain a stimulation of the mucosalimmune response.

The vaccine composition according to the invention can advantageously beprovided in various pharmaceutical formulations which are particularlywell suited to the route of administration and to the desired effect,i.e. obtaining a humoral, cellular and/or mucosal immune response.

A subject of the present invention is thus also a pharmaceuticalformulation intended for oral administration, characterized in that itessentially consists of:

a core consisting of a vaccine composition as defined above embedded ina gelatin and

a coating selected from the group consisting of a film-forming polymerwhich is soluble or expandable in water and soluble in solvents andwhich is selected from the group consisting of cellulose derivatives,polyvinylpyrrolidone, acrylic and methacrylic esters, polyethyleneglycols, polyvinyl alcohols, vinylpyrrolidone/vinyl acetate copolymer,vinylpyrrolidone/polyvinyl alcohol copolymer and protein substances suchas zein or gliadin.

Preferably, said film-forming agent is selected from the groupconsisting of cellulose ethers and esters, such as cellulose acetate,cellulose acetate phthalate, cellulose butyrate, ethylcellulose andmethylcellulose.

According to one advantageous embodiment of said formulation, saidfilm-forming polymer is combined with at least one plasticizer chosenfrom glycerol and esters thereof, high molecular weightpolyethyleneglycols, ricin oil and citric, phthalic, adipic and sebacicacid esters.

Such a formulation, which is intended for oral administration, protectsthe recombinant Env protein (antigen) from degradation by gastricproteases and from the acid pH of the stomach. The coating dissolves inthe alkaline pH of the intestine, which releases the antigen in thevicinity of Peyer's patches, which are the major sites of induction ofmucosal immunity.

According to another advantageous embodiment of said formulation, saidvaccine composition consists of a freeze-dried mixture of immunosomes,onto which a gp120/160 protein is anchored, with trehalose.

A preferred formulation intended to be administered orally comprises:

a core consisting of a freeze-dried mixture of immunosomes onto which agp120/160 protein is anchored and of trehalose, embedded in gelatin and

a coating consisting of a cellulose derivative, preferably celluloseacetate phthalate.

A subject of the present invention is thus also a pharmaceuticalformulation intended for local administration to a mucous membrane(vaginal or rectal), characterized in that it essentially consists of avaccine composition, as defined, above embedded in glycerol or aglycerol/glycerine-based mixture.

According to one advantageous embodiment of said formulation, saidvaccine composition consists of a freeze-dried mixture of immunosomes,onto which a gp120/160 protein is anchored, with trehalose.

Besides the above arrangements, the invention comprises yet otherarrangements which will emerge from the description which follows, whichrefers to examples of implementation of the method which is the subjectof the present invention.

It should be clearly understood, however, that these examples are givenonly by way of illustration of the subject of the invention, of whichthey in no way constitute a limitation.

EXAMPLE 1 Preparation of the EnvΔV3-GPGRAF Recombinant Protein

Partial Deletion of the V3 Loop

The env gene of HIV-1_(LAI) is cloned into a baculoviral system; thevariable sequences of the V3 loop were eliminated by introducing amodification into the env gene of pNL4-3, conserving only thenucleotides encoding the GPGRAF (SEQ ID NO: 1) hexapeptide and the twocysteines at the base of the loop.

This modification of the V3 loop was carried out with the aid of 4oligonucleotides. They were hybridized so as to reconstitute themodified V3 loop and cloned directly between the Ase1 and Nhe1restriction sites of an intermediate vector comprising the first 1035nucleotides of the env gene, in such a way as to conserve only theGPGRAF (SEQ ID NO: 1) motif and the two cysteines of the V3 region.

The modification introduced into the gene was confirmed by sequencingthe V3 region, and cloned into the previously constructed transfervector pBacPAK env (7) in order to obtain the transfer vector pBacPAKenvΔV3-GPGRAF. The latter made it possible to generate the envΔV3-GPGRAFrecombinant baculovirus (7).

Expression of the Recombinant Env Gene

Sf21 insect cells were infected with the Autographa californica nuclearpolyhedrosis virus (AcNPV), as well as with the envΔV3-GPGRAFrecombinant baculovirus. Three to four days post-infection, the cellswere harvested, lysed in the presence of detergent and analysed byelectrophoresis on 10% polyacrylamide gel, as well as by Western blot.The results showed that the cells infected with the envΔV3-GPGRAFrecombinant baculovirus express a protein which has a molecular masscompatible with deletion. This protein is recognized by a humanreference serum which is positive for the HIV-1 antigens (7).

Purification of ΔV3-GPGRAF Recombinant gp160

EnvΔV3-GPGRAF recombinant gp160 was purified by chromatography on aDEAE-cellulose column, followed by a purification on a Lens culinarislectin column, from 2×10⁹ Sf21 cells infected with the envΔV3-GPGRAFrecombinant baculovirus. Analysis by electrophoresis showed that theprotein was more than 80% pure.

EXAMPLE 2 Preparation of a Pharmaceutical Formulation According to theInvention

a. Preparation of Immunosomes with ΔV3-GPGRAF Recombinant gp160

The immunosomes were prepared by anchoring ΔV3-GPGRAF recombinant gp160onto preformed liposomes in accordance with the method described inpatent EP 47 480. The ΔV3-GPGRAF-immunosomes are particles ofapproximately 90 nm which are covered with ΔV3-GPGRAF-gp160.

b. Formulations of the Composition Obtained in a.

For the oral immunizations, the ΔV3-GPGRAF-immunosomes are freeze-driedin the presence of threalose, and the antigen is introduced into agelatin capsule. The capsule is coated with a mixture containingcellulose acetate phthalate, which protects the antigen from degradationby gastric proteases and from the acid pH of the stomach. The coatingdissolves in the alkaline pH of the intestine, which releases theantigen in the vicinity of Peyer's patches, which are the major sites ofinduction of mucosal immunity.

For the immunizations via the vaginal or rectal route, the antigen isformulated in a glycerol/glycerine-based mixture which is solid at roomtemperature but which melts at physiological body temperature, thusgradually releasing the antigen.

EXAMPLE 3 Demonstration of the Immunogenic and Vaccine Activity of aFormulation According to Example 2

Protocol for Immunization of C57/BL Mice

In this hyperimmunization protocol, 12 mice received four injections ofimmunosomes containing 25 μg of ΔV3-GPGRAF-gp160 intraperitoneally at3-week intervals, followed by an intravenous booster. No adjuvant wasused. Six control mice were subjected to the same protocol using PBS.

Evaluation by ELISA of the Immune Response Against the LAI, IIIB, MN andRF Strains

Two weeks after the intravenous booster, the mice were bled byintracardiac puncture, and the sera were evaluated for the presence ofIgM, IgG and IgA antibodies which react with the LAI, IIIB, MN and RFstrains.

All the mice developed very high titres of IgG-type antibodies againsteach of the four strains tested, which were between 1/65 536 and 1/524288. Tables I, II, III and IV show that the mice also developedantibodies of the three isotypes against four laboratory strains tested.TABLE I Humoral immune response of mice immunized with animmunosome-anchored ΔV3-GPGRAF-gp160 composition Titre by ELISA ofantibodies Immuni- directed against the LAI strain Mouse zations AntigenIgM IgG IgA 1 SIX IMS-ΔV3- 1/2 048 1/262 144 1/256 GPGRAF (25 μg) 2 ″IMS-ΔV3- 1/1 024 1/262 144 1/256 GPGRAF (25 μg) 3 ″ IMS-ΔV3- 1/2 0481/524 288 1/512 GPGRAF (25 μg) 4 ″ IMS-ΔV3-  1/512 1/131 072 1/256GPGRAF (25 μg) 5 ″ IMS-ΔV3- 1/1 024 1/131 072 1/128 GPGRAF (25 μg) 6 ″IMS-ΔV3- 1/2 048 1/262 144 1/256 GPGRAF (25 μg) 7 ″ IMS-ΔV3- 1/1 0241/262 144 1/256 GPGRAF (25 μg) 8 ″ IMS-ΔV3- 1/1 024  1/65 538 1/128GPGRAF (25 μg) 9 ″ IMS-ΔV3- 1/4 096 1/524 288 1/1 024 GPGRAF (25 μg) 10″ IMS-ΔV3- 1/4 096 1/524 288 1/1 024 GPGRAF (25 μg) 11 ″ IMS-ΔV3- 1/2048 1/262 144 1/256 GPGRAF (25 μg) 12 ″ IMS-ΔV3- 1/2 048 1/262 144 1/256GPGRAF (25 μg) 13 SIX PBS <1/32 <1/32 <1/32  14 ″ ″ <1/32 <1/32 <1/32 15 ″ ″ <1/32 <1/32 <1/32  16 ″ ″ <1/32 <1/32 <1/32  17 ″ ″ <1/32 <1/32<1/32  18 ″ ″ <1/32 <1/32 <1/32 

TABLE II Humoral immune response of mice immunized with animmunosome-anchored ΔV3-GPGRAF-gp160 composition Titre by ELISA ofantibodies Immuni- directed against the IIIB strain Mouse zationsAntigen IgM IgG IgA 1 SIX IMS-ΔV3- 1/2 048 1/262 144 1/256 GPGRAF (25μg) 2 ″ IMS-ΔV3- 1/1 024 1/262 144 1/256 GPGRAF (25 μg) 3 ″ IMS-ΔV3- 1/2048 1/262 144 1/512 GPGRAF (25 μg) 4 ″ IMS-ΔV3- 1/1 024 1/131 072 1/256GPGRAF (25 μg) 5 ″ IMS-ΔV3- 1/1 024 1/131 072 1/128 GPGRAF (25 μg) 6 ″IMS-ΔV3- 1/2 048 1/131 072 1/256 GPGRAF (25 μg) 7 ″ IMS-ΔV3- 1/1 0241/262 144 1/256 GPGRAF (25 μg) 8 ″ IMS-ΔV3- 1/1 024  1/65 536 1/128GPGRAF (25 μg) 9 ″ IMS-ΔV3- 1/4 096 1/131 072 1/1 024 GPGRAF (25 μg) 10″ IMS-ΔV3- 1/4 096 1/131 072 1/1 024 GPGRAF (25 μg) 11 ″ IMS-ΔV3- 1/2048 1/262 144 1/256 GPGRAF (25 μg) 12 ″ IMS-ΔV3- 1/2 048 1/131 072 1/256GPGRAF (25 μg) 13 SIX PBS <1/32 <1/32 <1/32  14 ″ ″ <1/32 <1/32 <1/32 15 ″ ″ <1/32 <1/32 <1/32  16 ″ ″ <1/32 <1/32 <1/32  17 ″ ″ <1/32 <1/32<1/32  18 ″ ″ <1/32 <1/32 <1/32 

TABLE III Humoral immune response of mice immunized with animmunosome-anchored ΔV3-GPGRAF-gp160 composition Titre by ELISA ofantibodies Immuni- directed against the MN strain Mouse zations AntigenIgM IgG IgA 1 SIX IMS-ΔV3- 1/2 048 1/131 072 1/256 GPGRAF (25 μg) 2 ″IMS-ΔV3- 1/1 024 1/131 072 1/256 GPGRAF (25 μg) 3 ″ IMS-ΔV3- 1/2 0481/131 072 1/512 GPGRAF (25 μg) 4 ″ IMS-ΔV3- 1/1 024  1/65 536 1/256GPGRAF (25 μg) 5 ″ IMS-ΔV3- 1/1 024  1/65 536 1/128 GPGRAF (25 μg) 6 ″IMS-ΔV3- 1/2 048  1/65 536 1/256 GPGRAF (25 μg) 7 ″ IMS-ΔV3- 1/1 0241/131 072 1/256 GPGRAF (25 μg) 8 ″ IMS-ΔV3- 1/1 024  1/65 536 1/128GPGRAF (25 μg) 9 ″ IMS-ΔV3- 1/4 096 1/131 072 1/1 024 GPGRAF (25 μg) 10″ IMS-ΔV3- 1/4 096 1/131 072 1/1 024 GPGRAF (25 μg) 11 ″ IMS-ΔV3- 1/2048 1/262 144 1/256 GPGRAF (25 μg) 12 ″ IMS-ΔV3- 1/2 048 1/131 072 1/256GPGRAF (25 μg) 13 SIX PBS <1/32 <1/32 <1/32  14 ″ ″ <1/32 <1/32 <1/32 15 ″ ″ <1/32 <1/32 <1/32  16 ″ ″ <1/32 <1/32 <1/32  17 ″ ″ <1/32 <1/32<1/32  18 ″ ″ <1/32 <1/32 <1/32 

TABLE IV Humoral immune response of mice immunized with animmunosome-anchored ΔV3-GPGRAF-gp160 composition Titre by ELISA ofantibodies Immuni- directed against the RF strain Mouse zations AntigenIgM IgG IgA 1 SIX IMS-ΔV3- 1/1 024 1/131 072 1/128 GPGRAF (25 μg) 2 ″IMS-ΔV3- 1/1 024 1/131 072 1/128 GPGRAF (25 μg) 3 ″ IMS-ΔV3- 1/2 0481/131 072 1/256 GPGRAF (25 μg) 4 ″ IMS-ΔV3- 1/1 024  1/65 536 1/128GPGRAF (25 μg) 5 ″ IMS-ΔV3- 1/1 024  1/65 536 1/64  GPGRAF (25 μg) 6 ″IMS-ΔV3- 1/2 048  1/65 536 1/126 GPGRAF (25 μg) 7 ″ IMS-ΔV3- 1/1 0241/131 072 1/126 GPGRAF (25 μg) 8 ″ IMS-ΔV3- 1/1 024  1/65 536 1/64 GPGRAF (25 μg) 9 ″ IMS-ΔV3- 1/1 024 1/131 072 1/256 GPGRAF (25 μg) 10 ″IMS-ΔV3- 1/2 048 1/131 072 1/256 GPGRAF (25 μg) 11 ″ IMS-ΔV3- 1/2 0481/262 144 1/128 GPGRAF (25 μg) 12 ″ IMS-ΔV3- 1/1 024 1/131 072 1/128GPGRAF (25 μg) 13 SIX PBS <1/32 <1/32 <1/32  14 ″ ″ <1/32 <1/32 <1/32 15 ″ ″ <1/32 <1/32 <1/32  16 ″ ″ <1/32 <1/32 <1/32  17 ″ ″ <1/32 <1/32<1/32  18 ″ ″ <1/32 <1/32 <1/32 

Determination of the Presence of Antibodies Which are Capable ofNeutralizing the Infectivity of Different Laboratory Strains

The sera of mice immunized with the ΔV3-GPGRAF-gp160 immunosome werethen evaluated for their potential for neutralizing the infectivity ofthe LAI, IIIB, MN, RF, LAV 43.01 and BAL strains. The neutralizationassays are carried out using CEM cells. All the mice developedneutralizing antibodies ranging from 1/1024 to 1/126, as illustrated inTables V and VI. TABLE V Titre of neutralizing antibodies directedagainst divergent strains of HIV-1 in mice immunized with theimmunosome-anchored ΔV3-GPGRAF-gp160 vaccine composition Titre ofantibodies which neu- Immuni- tralize against 100 TCID₅₀ of: Mousezations Antigen LAI IIIB RF 1 SIX IMS-ΔV3- 1/256 1/256 1/256 GPGRAF (25μg) 2 ″ IMS-ΔV3- 1/256 1/256 1/256 GPGRAF (25 μg) 3 ″ IMS-ΔV3- 1/5121/256 1/512 GPGRAF (25 μg) 4 ″ IMS-ΔV3- 1/256 1/128 1/256 GPGRAF (25 μg)5 ″ IMS-ΔV3- 1/256 1/256 1/128 GPGRAF (25 μg) 6 ″ IMS-ΔV3- 1/256 1/1261/256 GPGRAF (25 μg) 7 ″ IMS-ΔV3- 1/1 024 1/256 1/256 GPGRAF (25 μg) 8 ″IMS-ΔV3- 1/64  1/32  1/128 GPGRAF (25 μg) 9 ″ IMS-ΔV3- 1/256 1/256 1/1024 GPGRAF (25 μg) 10 ″ IMS-ΔV3- 1/256 1/126 1/1 024 GPGRAF (25 μg) 11 ″IMS-ΔV3- 1/512 1/512 1/256 GPGRAF (25 μg) 12 ″ IMS-ΔV3- 1/256 1/1261/256 GPGRAF (25 μg) 13 SIX PBS <1/32  <1/32  <1/32  14 ″ ″ <1/32 <1/32  <1/32  15 ″ ″ <1/32  <1/32  <1/32  16 ″ ″ <1/32  <1/32  <1/32  17″ ″ <1/32  <1/32  <1/32  18 ″ ″ <1/32  <1/32  <1/32 

TABLE VI Titre of neutralizing antibodies directed against divergentstrains of HIV-1 in mice immunized with the immunosome-anchoredΔV3-GPGRAF-gp160 vaccine composition Titre of antibodies which neu-tralize against 100 TCID₅₀ of: Immuni- LAV Mouse zations Antigen 43.01MN BAL 1 SIX IMS-ΔV3- 1/256 1/126 1/126 GPGRAF (25 μg) 2 ″ IMS-ΔV3-1/256 1/126 1/64  GPGRAF (25 μg) 3 ″ IMS-ΔV3- 1/1 024 1/256 1/256 GPGRAF(25 μg) 4 ″ IMS-ΔV3- 1/256 1/128 1/126 GPGRAF (25 μg) 5 ″ IMS-ΔV3- 1/2561/256 1/128 GPGRAF (25 μg) 6 ″ IMS-ΔV3- 1/256 1/126 1/256 GPGRAF (25 μg)7 ″ IMS-ΔV3- 1/1 024 1/256 1/256 GPGRAF (25 μg) 8 ″ IMS-ΔV3- 1/32  1/32 1/64  GPGRAF (25 μg) 9 ″ IMS-ΔV3- 1/256 1/256 1/256 GPGRAF (25 μg) 10 ″IMS-ΔV3- 1/256 1/126 1/126 GPGRAF (25 μg) 11 ″ IMS-ΔV3- 1/1 024 1/5121/256 GPGRAF (25 μg) 12 ″ IMS-ΔV3- 1/256 1/126 1/126 GPGRAF (25 μg) 13SIX PBS <1/32  <1/32  <1/32  14 ″ ″ <1/32  <1/32  <1/32  15 ″ ″ <1/32 <1/32  <1/32  16 ″ ″ <1/32  <1/32  <1/32  17 ″ ″ <1/32  <1/32  <1/32  18″ ″ <1/32  <1/32  <1/32 

Evaluation of the Neutralizing Power of the Sera Against Six PrimaryIsolates

Finally, the neutralizing power of the mouse sera was determined againstsix primary isolates: 03908, 65869, 65965, 65870, 65871 and 3929,generated from coculture of lymphocytes from patients at various stagesof the disease, with lymphocytes from seronegative donors. Theneutralization assays were carried out using non-stimulated PBLs. Allthe mice developed antibodies which were capable of neutralizing theinfectivity of primary isolates. By way of example, see Tables VII andVIII. The titres were generally very high, these titres being between1/512 and 1/256 against five of the six primary isolates tested. Isolate65869 proved to be more resistant to neutralization. The titres were1/64 and 1/32 and <1/32 in four of the sera. This isolate came from apatient in the terminal phase of the disease, and the virus inducedgigantic syncytia in the cell cultures. TABLE VII Titre of neutralizingantibodies directed against divergent strains of HIV-1 in mice immunizedwith the immunosome-anchored ΔV3-GPGRAF-gp160 vaccine composition Titreof antibodies which neu- Immuni- tralize against 100 TCID₅₀ of: Mousezations Antigen # 03908 # 65869 # 65965 1 SIX IMS-ΔV3- 1/126 <1/32 1/64 GPGRAF (25 μg) 2 ″ IMS-ΔV3- 1/64   1/32 1/126 GPGRAF (25 μg) 3 ″IMS-ΔV3- 1/256  1/32 1/126 GPGRAF (25 μg) 4 ″ IMS-ΔV3- 1/126 <1/32 1/256GPGRAF (25 μg) 5 ″ IMS-ΔV3- 1/256  1/64 1/126 GPGRAF (25 μg) 6 ″IMS-ΔV3- 1/512  1/32 1/512 GPGRAF (25 μg) 7 ″ IMS-ΔV3- 1/256  1/64 1/256GPGRAF (25 μg) 8 ″ IMS-ΔV3- <1/32  <1/32 <1/32  GPGRAF (25 μg) 9 ″IMS-ΔV3- 1/512 <1/32 1/256 GPGRAF (25 μg) 10 ″ IMS-ΔV3- 1/256  1/321/126 GPGRAF (25 μg) 11 ″ IMS-ΔV3- 1/512  1/32 1/256 GPGRAF (25 μg) 12 ″IMS-ΔV3- 1/256  1/32 1/126 GPGRAF (25 μg) 13 SIX PBS <1/32  <1/32 <1/32 14 ″ ″ <1/32  <1/32 <1/32  15 ″ ″ <1/32  <1/32 <1/32  16 ″ ″ <1/32 <1/32 <1/32  17 ″ ″ <1/32  <1/32 <1/32  18 ″ ″ <1/32  <1/32 <1/32 

TABLE VIII Titre of neutralizing antibodies directed against divergentstrains of HIV-1 in mice immunized with the immunosome-anchoredΔV3-GPGRAF-gp160 vaccine composition Titre of antibodies which neu-Immuni- tralize against 100 TCID₅₀ of: Mouse zations Antigen # 65870 #65871 # 3929 1 SIX IMS-ΔV3- 1/64  1/126 1/64  GPGRAF (25 μg) 2 ″IMS-ΔV3- 1/126 1/126 1/126 GPGRAF (25 μg) 3 ″ IMS-ΔV3- 1/126 1/256 1/256GPGRAF (25 μg) 4 ″ IMS-ΔV3- 1/256 1/128 1/126 GPGRAF (25 μg) 5 ″IMS-ΔV3- 1/256 1/126 1/126 GPGRAF (25 μg) 6 ″ IMS-ΔV3- 1/256 1/64  1/256GPGRAF (25 μg) 7 ″ IMS-ΔV3- 1/126 1/256 1/256 GPGRAF (25 μg) 8 ″IMS-ΔV3- <1/32  <1/32  <1/32  GPGRAF (25 μg) 9 ″ IMS-ΔV3- 1/256 1/2561/256 GPGRAF (25 μg) 10 ″ IMS-ΔV3- 1/256 1/126 1/126 GPGRAF (25 μg) 11 ″IMS-ΔV3- 1/512 1/512 1/256 GPGRAF (25 μg) 12 ″ IMS-ΔV3- 1/256 1/1261/126 GPGRAF (25 μg) 13 SIX PBS <1/32  <1/32  <1/32  14 ″ ″ <1/32 <1/32  <1/32  15 ″ ″ <1/32  <1/32  <1/32  16 ″ ″ <1/32  <1/32  <1/32  17″ ″ <1/32  <1/32  <1/32  18 ″ ″ <1/32  <1/32  <1/32 

These results show that partially deleting the V3 loop while keeping theconserved sequence GPGRAF (SEQ ID NO: 1) promotes the induction ofwide-ranging antibodies which are capable of neutralizing variouslaboratory strains, but also various primary isolates.

Similar results are obtained with the protein which contains a totaldeletion of the V3 loop.

REFERENCES

-   (1) Emini E., Schleif W., Numberg, J. et al. 1992. Prevention of    HIV-1 infection in chimpanzees by gp120 V3 domain-specific    monoclonal antibody. Nature 355:728-30.-   (2) Girard M. P., Kieny M., Pinter A., et al. 1991. Immunization of    chimpanzees confers protection against challenge with human    immunodeficiency virus. Proc. Nat. Acad. Sci. USA 88:542-46.-   (3) Nara P. L., Garrity R. R., Goudsmit J. et al. 1991.    Neutralization of HIV-1: a paradox of humoral proportion. FASEB J.    5:2437-55.-   (4) Palker T. J., Claar M. E., Langlois A. J et al. 1988.    Type-specific neutralization of the human immunodeficiency virus    with antibodies to env-coded peptides. Proc. Nat. Acad. Sci. USA    85:1932-6.-   (5) Javaherian K., Langlois J., McDonald C. et al. 1989. Principal    neutralization domain of the human immunodeficiency virus type 1    envelope protein. Proc. Nat. Acad. Sci. USA 86:6768-72.-   (6) Lucinda A., Dubay J. W., Morris J. F. et al. 1992. V3 loop    region of the HIV-1 gp120 envelope protein is essential for virus    infectivity. Virology 187:423-32.-   (7) Lavallée Claude and Lise Thibodeau (1996) Clonage, expression et    caractérisation de gp160 du VIH-1, portant des délétions partielles    ou totales dans la boucle V3. [Cloning, expression and    characterization of HIV-1 gp160, bearing partial or total deletions    in the V3 loop] 1996 C.R. Acad.

Sci. Paris 319:983-990.

As emerges from the above, the invention is in no way limited to thoseof its modes of implementation, execution and application which havejust been described more explicitly; on the contrary, it embraces allthe variants thereof which may occur to persons skilled in the art,without departing from the context or the scope of the presentinvention.

1-12. (canceled)
 13. An immunogenic composition formulated for localadministration directly to a mucous membrane comprising a recombinantHIV-1 envelope protein comprising a mutated V3 loop, wherein the mutatedV3 loop comprises the GPGRAF (SEQ ID NO: 1) hexamer sequence flanked bythe two basal cysteines but lacks all or a portion of the rest of the V3loop, and said recombinant HIV-1 envelope protein being anchored ontopreformed liposomes, and wherein said immunogenic composition inducesproduction of neutralizing antibodies within a mammal that are capableof impeding infection of susceptible human cells by a plurality ofdifferent strains of HIV.
 14. The immunogenic composition of claim 13,wherein said immunogenic composition induces both a humoral, a cellular,and a mucosal immunity against HIV-1.